Robert C. Moschel

Phase I Trial of Temozolomide Plus O6-Benzylguanine for Patients With Recurrent or Progressive Malignant Glioma

PURPOSE We conducted a two-phase clinical trial in patients with progressive malignant glioma (MG). The first phase of this trial was designed to determine the dose of O6-BG effective in producing complete depletion of tumor AGT activity for 48 hours. The second phase of the trial was designed to define the maximum tolerated dose (MTD) of a single dose of temozolomide when combined with O6-BG. In addition, plasma concentrations of O6-BG and O6-benzyl-8-oxoguanine were evaluated after O6-BG. PATIENTS AND METHODS For our first phase of the clinical trial, patients were scheduled to undergo craniotomy for AGT determination after receiving a 1-hour O6-BG infusion at 120 mg/m2 followed by a continuous infusion at an initial dose of 30 mg/m2/d for 48 hours. The dose of the continuous infusion of O6-BG escalated until tumor AGT was depleted. Once the O6-BG dose was established a separate group of patients was enrolled in the second phase of clinical trial, in which temozolomide, administered as a single dose at the end of the 1-hour O6-BG infusion, was escalated until the MTD was determined. RESULTS The O6-BG dose found to be effective in depleting tumor AGT activity at 48 hours was an IV bolus of 120 mg/m2 over 1 hour followed by a continuous infusion of 30 mg/m2/d for 48 hours. On enrolling 38 patients in six dose levels of temozolomide, the MTD was established at 472 mg/m2 with dose-limiting toxicities limited to myelosuppression. CONCLUSION This study provides the foundation for a phase II trial of O6-BG plus temozolomide in temozolomide-resistant MG.

Phase I trial of O6-benzylguanine for patients undergoing surgery for malignant glioma.

PURPOSE The major mechanism of resistance to alkylnitrosourea therapy is the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT), which removes chlorethylation or methylation damage from the O6-position of guanine. O6-benzylguanine (O6-BG) is an AGT substrate that inhibits AGT by suicide inactivation. We conducted a phase I trial to define the presurgical dose required for depletion of tumor AGT activity in patients with malignant glioma. MATERIALS AND METHODS Patients were to be treated 18 hours before craniotomy with intravenous doses that ranged between 40 and 100 mg/m2 given over 1 hour. Resected tumor was snap-frozen in liquid nitrogen and AGT activity analyzed by high-pressure liquid chromatography (HPLC). Up to 13 patients were treated at a specific dose of O6-BG, with a target end point of > or = 11 of 13 patients with undetectable tumor AGT levels (< 10 fmol/mg protein). RESULTS Thirty patients with malignant gliomas were enrolled, with 11 of 11 patients treated at 100 mg/m2 O6-BG demonstrating tumor AGT levels less than 10 fmol/mg protein. No toxicity was noted in any patient treated. CONCLUSION These results indicate that 100 mg/m2 of O6-BG can maintain tumor AGT levels less than 10 fmol/mg protein for at least 18 hours after treatment, a time interval in which bis(2-chloroethyl)nitrosourea (BCNU)-induced chloroethyl adducts are fully converted into interstrand cross-links. A 100-mg/m2 dose of O6-BG will be used in combination with BCNU in another phase I trial designed to determine the maximal-tolerated dose of BCNU.

Phase II Trial of Carmustine Plus O6-Benzylguanine for Patients With Nitrosourea-Resistant Recurrent or Progressive Malignant Glioma

PURPOSE We conducted a phase II trial of carmustine (BCNU) plus the O(6)-alkylguanine-DNA alkyltransferase inhibitor O(6)-benzylguanine (O(6)-BG) to define the activity and toxicity of this regimen in the treatment of adults with progressive or recurrent malignant glioma resistant to nitrosoureas. PATIENTS AND METHODS Patients were treated with O(6)-BG at an intravenous dose of 120 mg/m(2) followed 1 hour later by 40 mg/m(2) of BCNU, with cycles repeated at 6-week intervals. RESULTS Eighteen patients were treated (15 with glioblastoma multiforme, two with anaplastic astrocytoma, and one with malignant glioma). None of the 18 patients demonstrated a partial or complete response. Two patients exhibited stable disease for 12 weeks before their tumors progressed. Three patients demonstrated stable disease for 6, 12, and 18 weeks before discontinuing therapy because of hematopoietic toxicity. Twelve patients experienced reversible > or = grade 3 hematopoietic toxicity. There was no difference in half-lives (0.56 +/- 0.21 hour v 0.54 +/- 0.20 hour) or area under the curve values (4.8 +/- 1.7 microg/mL/h v 5.0 +/- 1.3 microg/mL/h) of O(6)-BG for patients receiving phenytoin and those not treated with this drug. CONCLUSION These results indicate that O(6)-BG plus BCNU at the dose schedule used in this trial is unsuccessful in producing tumor regression in patients with nitrosourea-resistant malignant glioma, although stable disease was seen in five patients for 6, 12, 12, 12, and 18 weeks. Future use of this approach will require strategies to minimize dose-limiting toxicity of BCNU such as regional delivery or hematopoietic stem-cell protection.

Enhancement of Nitrosourea Activity in Medulloblastoma and Glioblastoma Multiforme

BACKGROUND Although chemotherapy offers promise of increased survival for children with medulloblastoma and glioblastoma multiforme, drug resistance occurs frequently, resulting in tumor progression and death. Resistance to nitrosoureas and methylating agents, which damage DNA, can be mediated by a DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGAT). Depletion of this protein with alkylguanines or methylating agents, however, restores tumor cell sensitivity to the cytotoxicity of chloroethylnitrosoureas (e.g., carmustine [BCNU]). PURPOSE This study was designed to determine whether resistance to the activity of nitrosourea (the drug BCNU) in BCNU-resistant human medulloblastoma (D341 Med) and human glioblastoma multiforme (D-456 MG) can be reversed by the methylating agent streptozocin and the O6-substituted guanines O6-methylguanine and O6-benzylguanine. METHODS Xenografts were grown subcutaneously in athymic BALB/c mice. BCNU was administered as a single intraperitoneal injection at doses of 100 mg/m2, 75 mg/m2, or 38 mg/m2--i.e., 1.0, 0.75, or 0.38, respectively, of the dose lethal to 10% of treated animals (LD10). Mice were treated intraperitoneally with a single dose of O6-benzylguanine or O6-methylguanine (240 mg/m2) or with streptozocin (600 mg/m2) daily for 4 days. Response was assessed by tumor growth delay and tumor regression. AGAT activity in the xenografts was measured at 1 and 6 hours after pretreatment, at the time tumors were excised. RESULTS Pretreatment with O6-benzylguanine, O6-methylguanine, or streptozocin reduced AGAT activity to 4%, 25%, and 95% of control values, respectively, in D341 Med and 0%, 0%, and 25% of control values, respectively, in D-456 MG 1 hour after injection. After 6 hours, levels changed to 7%, 61%, and 116% of control values in D341 Med and 0%, 79%, and 21% of control values in D-456 MG, respectively. Both D341 Med and D-456 MG xenografts were completely resistant to BCNU at its LD10. Pretreatment with O6-benzylguanine increased BCNU sensitivity in both types of xenograft. In contrast, treatment with BCNU plus O6-methylguanine or streptozocin did not produce growth delays substantially different from those produced by BCNU alone, reflecting the more efficient depletion of AGAT by O6-benzylguanine. Following therapy with BCNU plus O6-benzylguanine at 0.38 LD10, tumor regressions were seen in eight of 10 D341 Med and in all 10 D-456 MG xenografts. CONCLUSION We recommend comprehensive clinical toxicologic evaluation of combination therapy with O6-benzylguanine plus BCNU, which would allow subsequent design of phase I clinical trials.

Modulation of mammalian O6-alkylguanine-DNA alkyltransferase in vivo by O6-benzylguanine and its effect on the sensitivity of a human glioma tumor to 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea.

Experiments were carried out in mice and hamsters to determine whether the activity of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, in tissues and tumors was reduced by treatment with O6-benzylguanine in vivo. Following intraperitoneal injection of O6-benzylguanine, there was a rapid and complete loss of alkyltransferase activity in both livers and kidneys of mice and hamsters. The activity in mouse tissues was slowly restored, reaching pretreatment activities at 16 hr and 72 hr after injection of O6-benzylguanine at 10 mg/kg or 126 mg/kg, respectively. The activity in hamster liver was restored at a significantly lower rate, reaching less than 20% pretreatment activity 72 hr after treatment with 100 mg/kg of O6-benzylguanine. The efficient reduction of alkyltransferase activity by O6-benzylguanine was in sharp contrast to the inability of O6-methylguanine to bring about similar reductions. Activities dropped to about 55% of pretreatment activities in several mouse organs 4 hr after treatment with 126 mg/kg of O6-methylguanine compared to a more than 90% reduction in activity in animals after treatment with O6-benzylguanine. The sensitivity of SF767 cells to meCCNU after treatment with O6-benzylguanine was increased substantially. Furthermore, treatment of nude mice carrying SF767 tumor with 60 mg/kg of O6-benzylguanine prior to either 7.5 or 15 mg/kg of meCCNU led to significant inhibition of tumor growth. These studies indicate that O6-benzylguanine is a suitable compound for use in experiments to examine the role of the alkyltransferase protein in vivo in counteracting the effects of alkylating agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of subcutaneous and intracranial brain tumor xenografts withO6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea

O6-Alkylguanine-DNA alkyltransferase (AT) is a cellular protein that protects cells from the cytotoxic effects of nitrosoureas by repairing alkyl lesions at theO6 position of guanine. We have studied the ability ofO6-benzylguanine to deplete AT activity in brain tumor xenografts and thereby increase the sensitivity of these tumors to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). In toxicity studies, pretreatment of athymic mice withO6-benzylguanine increased the toxicity of BCNU significantly. After i.p. injection ofO6-benzylguanine into athymic mice carrying subcutaneous (s. c.) D341MED, a human medulloblastoma xenograft with a high AT activity, the AT activity of the tumors became undetectable within 1 h and remained depleted until 36 h. In s. c. xenografts to D341MED, treatment withO6-benzylguanine followed 1 h later by BCNU produced a significantly greater growth delay (14.8 days) than was seen with BCNU alone (2.3 days). A lower pretreatment dose ofO6-benzylguanine produced a significantly smaller therapeutic effect. Delaying the administration of BCNU until 36 h afterO6-benzylguanine resulted in a growth delay (1.2 days) that was not significantly different from that produced by the control or BCNU alone. In athymic mice with intracranial (i.c.) xenografts of D341MED, pretreatment withO6-benzylguanine followed 1 h later by BCNU produced a significantly increased survival as compared with that of the control, BCNU alone,O6-benzylguanine alone, andO6-benzylguanine followed 36 h later by BCNU. In experiments with s.c. xenografts of D245MG, a human glioma xenograft with undetectable AT activity, pretreatment withO6-benzylguanine 1 h prior to BCNU produced a significantly greater effect than was seen with BCNU treatment alone. The combination regimen, however, was not as effective as an equitoxic dose of BCNU alone. These studies suggest thatO6-benzylguanine may be a useful adjuvant to nitrosourea therapy in human malignancies that exhibit a range of AT activities and that dose and timing are important variables in achieving therapeutic success. These data also indicate that therapeutic potentiation of BCNU byO6-benzylguanine can be achieved in i.c. tumors. As a result, this approach may be useful in the treatment of neoplasms of the central nervous system.

Effect of O6-benzylguanine on the sensitivity of human colon tumor xenografts to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU).

A number of trials were conducted to determine the effect of O6-benzylguanine pretreatment on the sensitivity of human colon tumor xenografts to the antitumor effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). O6-Benzylguanine has been shown to inactivate the DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), which is primarily responsible for resistance to alkylnitrosoureas including BCNU. Colon tumor xenografts carried in nude mice were analyzed for their AGT content, and tumors with low, intermediate and high levels were chosen for further study. The AGT activity of HC-1, GC-3, VRC-5 and CX-1 human colon tumor xenografts was 16, 113, 180 and 367 fmol/mg protein, respectively. Treatment of mice consisted of vehicle alone, 6.25 to 50 mg/kg BCNU administered alone or BCNU (6.25 to 25 mg/kg) 1 hr after 120 mg/kg O6-benzylguanine on days 7 and 14 post-inoculation. Toxicity studies revealed that pretreatment with O6-benzylguanine increased the toxicity of BCNU, requiring administration of about 4-fold less drug. The growth of the VRC-5 tumor at day 42 post-inoculation was inhibited by 39% following treatment with 12.5 mg/kg BCNU alone and 92% when BCNU was combined with O6-benzylguanine pretreatment. The combination of O6-benzylguanine and BCNU (12.5 mg/kg) at day 42 resulted in an inhibition of HC-1 and CX-1 tumor growth by 84 and 72%, whereas BCNU alone inhibited growth by 54 and 14%, respectively. Therefore, the degree to which the antitumor effect of BCNU was increased by O6-benzylguanine pretreatment was dependent on the AGT activity, with a greater effect in tumors of intermediate or high activity. These data suggest that there is a role for O6-benzylguanine combined with BCNU in the treatment of human colon tumors.

Modulation of cyclophosphamide activity by O 6-alkylguanine-DNA alkyltransferase

Purpose: The human medulloblastoma cell line D283 Med (4-HCR), a line resistant to 4-hydroperoxycyclophosphamide (4-HC), displays enhanced␣repair of DNA interstrand crosslinks induced by phosphoramide mustard. D283 Med (4-HCR) cells are cross-resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea, but partial sensitivity is restored after elevated levels of O6-alkylguanine-DNA alkyltransferase (AGT) are depleted by O6-benzylguanine (O6-BG). Studies were conducted to define the activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide against D283 Med (4-HCR) after AGT is depleted by O6-BG. Methods: Limiting dilution and xenograft studies were conducted to define the activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide with or without O6-BG. Results: The activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide against D283 Med (4-HCR) was increased after AGT depletion by O6-BG preincubation. Similar studies with Chinese hamster ovary cells, with or without stable transfection with a plasmid expressing the human AGT protein, revealed that the AGT-expressing cells were significantly less sensitive to 4-HC and 4-hydroperoxydidechlorocyclophosphamide. Reaction of DNA with 4-HC, phosphoramide mustard, or acrolein revealed that only 4-HC and acrolein caused a decrease in AGT levels. Conclusions: We propose that a small but potentially significant part of the cellular toxicity of cyclophosphamide in these cells is due to acrolein, and that this toxicity is abrogated by removal of the acrolein adduct from DNA by AGT.

Repair of O6-Benzylguanine by the Escherichia coli Ada and Ogt and the Human O6-Alkylguanine-DNA Alkyltransferases

O6-Methylguanine is removed from DNA via the transfer of the methyl group to a cysteine acceptor site present in the DNA repair protein O6-alkylguanine-DNA alkyltransferase. The human alkyltransferase is inactivated by the free base O6-benzylguanine, raising the possibility that substantially larger alkyl groups could also be accepted as substrates. However, the Escherichia coli alkyltransferase, Ada-C, is not inactivated by O6-benzylguanine. The Ada-C protein was rendered capable of reaction by the incorporation of two site-directed mutations converting Ala316 to a proline (A316P) and Trp336 to alanine (W336A) or glycine (W336G). These changes increase the space at the active site of the protein where Cys321 is buried and thus permit access of the O6-benzylguanine inhibitor. Reaction of the mutant A316P/W336A-Ada-C with O6-benzylguanine was greatly stimulated by the presence of DNA, providing strong support for the concept that binding of DNA to the Ada-C protein activates the protein. The Ada-C protein was able to repair O6-benzylguanine in a 16-mer oligodeoxyribonucleotide. However, the rate of repair was very slow, whereas the E. coli Ogt, the human alkyltransferase, and the mutant A316P/W336A-Ada-C alkyltransferases reacted very rapidly with this 16-mer substrate and preferentially repaired it when incubated with a mixture of the methylated and benzylated 16-mers. These results show that benzyl groups are better substrates than methyl groups for alkyltransferases provided that steric factors do not prevent binding of the substrate in the correct orientation for alkyl group transfer.

Human liver oxidative metabolism of O6-benzylguanine

The oxidation of O6-benzylguanine, an inactivator of O6-alkylguanine-DNA alkyltransferase, was examined using human liver cytosol, microsomes, and several P450 isoforms. Incubation of O6-benzylguanine with human liver cytosol resulted in the formation of O6-benzyl-8-oxoguanine, which was inhibited by menadione, a potent inhibitor of aldehyde oxidase. Inhibition by allopurinol, a xanthine oxidase inhibitor, was less dramatic. Oxidation of O6-benzylguanine also occurred with pooled human liver microsomes and was inhibited by both furafylline and troleandomycin, selective inhibitors of CYP1A2 and CYP3A4, respectively. Human P450s CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2E1, and CYP3A4 expressed in Hep G2 hepatoma cells using vaccinia virus vectors were incubated with 10 or 200 microM O6-benzylguanine. At 10 microM, O6-benzylguanine was oxidized primarily by CYP1A2 and to a lesser extent by CYP3A4. However, an appreciable increase in CYP3A4 contribution was noted at 200 microM. CYP1A2 exhibited a more than 200-fold higher relative catalytic activity (Vmax/Km) compared with CYP3A4. Therefore, at therapeutically relevant concentrations of O6-benzylguanine, CYP1A2 could be primarily involved in its oxidation since it shows a much lower Km value (1.3 microM) than CYP3A4 (52.2 microM) and cytosol (81.5 microM). However, one would expect interindividual variation in the extent of oxidation of O6-benzylguanine depending on the levels of aldehyde oxidase, CYP1A2, and CYP3A4.